Human papillomaviruses (HPVs) infect mucoasal and cutaneous epithelium. About 6.2M new cases of sexually transmitted HPV infections are reported every year, and >20M people in the US are currently infected. HPV infections cause anogential cancers, especially cervical cancer, and are linked to >99% of all cervical malignancies. HPV types 16 and 31 cause anogenital infections and related cancers, but each causes a distinct infectious outcome and the viruses have different biological characteristics. Major obstacles in HPV research have included the lack of high-titer infectious viral stocks and the lack of an in vitro assay system for the study of early infection phases and viral gene expression. We have been successful in generating high-titer HPV virion stocks and we show evidence that oncogenic HPV types HPV16 and HPV31 interact differently with normal host cells, human keratinocytes (HKs). HPV16 has an internalization half time (t1/2) of ~4h, whereas the t1/2 for HPV31 is ~14h; HPV16 enters via clathrin-coated pits, but HPV31 uses a clathrin-independent, caveolae- and dynamin 2-dependent pathway in HKs that involves lipid rafts. Unlike previous studies, we will investigate bona fide HPV infections in normal host HKs. We will use a combination of multi-parameter confocal and video microscopic visualization (localization) and infectious entry assays (detection of viral transcription) to assess the routes of infection of normal host cells. Our central hypothesis is that specific external viral capsid features dictate the dissimilarities in the early infection events of HPV16 and HPV31 in HK. We will exploit the differences between HPV16 and HPV31 to pursue this program's goals, which are to identify the viral and cellular factors and define the mechanisms dictating the viral infection process in host HKs. To test our hypothesis, we will pursue three inter-related Specific Aims: In Aim 1 we will determine the viral and cellular factors affecting initial interactions with HKs. This will employ mixed virions comprised of HPV16 L1+HPV31 L2 (or HPV31L1+HPV16L2) and video microscopy of single particle tracking on host cells. In Aim 2 we will determine the requirement for intracellular signaling in virion entry and trafficking. In Aim 3 we will use novel dual fluor-labeled infectious particles created in our lab to delineate the route of intracellular trafficking and the location of virion uncoating. These studies will reveal the intracellular vesicles involved, and investigate the role of L2 in vesicle escape. We are employing established techniques for studying virus internalization and trafficking, now possible for HPVs due to the availability of high-titer viral stocks. This work will provide a foundation for defining the molecular mechanisms that control the establishment of HPV infections. Understanding the processes of viral entry will impact future development of broadly cross-protective prophylactic strategies for preventing persistent oncogenic HPV infections, a major risk for morbidity and malignant progression. Our findings will also provide significant insight into receptor-mediated endocytosis in keratinocytes, an area vastly understudied.